Bottom Line:
MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition.In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9.Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.

ABSTRACTThe Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.

Mentions:
Next, we examined alterations in mitochondria-associated proteins as an indication of disturbance of mitochondria that could be induced by CRISPR/Cas9-mediated mtDNA cleavage. When mtDNA was truncated at the Cox1 and Cox3 loci by CRISPR/Cas9, transcription of mtDNA heavy strands and thus expression of downstream genes from Cox1 locus could be affected (Figure 1(b)). When we monitored several mitochondrial marker proteins, there were alterations in several proteins: the levels of SDHA, heat shock protein 60 (HSP60), and prohibitin 1 (PHB1) decreased, whereas pyruvate dehydrogenase (PDH) and superoxide dismutase 1 (SOD1) levels increased (Figures 2(a) and 2(b)). However, no significant changes were observed in CoxIV or Cytochrome C (CytC) (Figures 2(a) and 2(b)).

Mentions:
Next, we examined alterations in mitochondria-associated proteins as an indication of disturbance of mitochondria that could be induced by CRISPR/Cas9-mediated mtDNA cleavage. When mtDNA was truncated at the Cox1 and Cox3 loci by CRISPR/Cas9, transcription of mtDNA heavy strands and thus expression of downstream genes from Cox1 locus could be affected (Figure 1(b)). When we monitored several mitochondrial marker proteins, there were alterations in several proteins: the levels of SDHA, heat shock protein 60 (HSP60), and prohibitin 1 (PHB1) decreased, whereas pyruvate dehydrogenase (PDH) and superoxide dismutase 1 (SOD1) levels increased (Figures 2(a) and 2(b)). However, no significant changes were observed in CoxIV or Cytochrome C (CytC) (Figures 2(a) and 2(b)).

Bottom Line:
MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition.In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9.Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.

ABSTRACTThe Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.